To link to the entire object, paste this link in email, IM or documentTo embed the entire object, paste this HTML in websiteTo link to this page, paste this link in email, IM or documentTo embed this page, paste this HTML in website

STRUCTURE AND FUNCTION OF ARCHAEAL MCM HELICASE
by
Aaron Samuel Brewster
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(MOLECULAR BIOLOGY)
May 2010
Copyright 2010 Aaron Samuel Brewster

The mini-chromosome maintenance protein (MCM) complex is an essential helicase for DNA replication in Archaea and Eukaryotes. While the eukaryotic complex consists of six homologous proteins (MCM2-7), the archaeon Sulfolobus solfataricus has only one MCM protein (ssoMCM), six subunits of which form a homohexamer. Here, I first discuss my 4.35Å crystal structure of the near full-length ssoMCM. The structure shows an elongated fold, with five sub-domains that are organized into two large N- and C-terminal domains. A near full-length ssoMCM hexamer generated based on the 6-fold symmetry of the N-terminal Methanothermobacter thermautotrophicus (mtMCM) hexamer shows inter-subunit distances suitable for bonding contacts, including the interface around the ATP pocket. Four unusual beta-hairpins of each subunit are located inside the central channel or around the side channels in the hexamer. Additionally, the hexamer fits well into the double-hexamer EM map of mtMCM. Mutational analysis of residues at the inter-subunits interface and around the side channels demonstrates their critical roles for hexamerization and helicase function. I also present a series of 25 structure based mutations, 9 of 10 of which have been characterized by DNA binding, ATPase and helicase assays. Finally, modeling of ATP binding and hydrolysis based on the above evidence is presented.

STRUCTURE AND FUNCTION OF ARCHAEAL MCM HELICASE
by
Aaron Samuel Brewster
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(MOLECULAR BIOLOGY)
May 2010
Copyright 2010 Aaron Samuel Brewster